Bacterial type IV secretion systems are a functionally diverse family of secretion systems present in many species of bacteria. These systems translocate DNA and/or protein substrates into bacteria and eukaryotes. T4SSs are composed of two large subfamilies, the conjugation machines and the effector translocators. T4SSs can also be subdivided based on sequence similarity of the components of their secretion apparatus. Type IVA secretion systems (T4ASSs) include related plasmids (e.g. F and RP4) and the Agrobacterium tumefaciens VirB system. T4BSSs comprise the Dot/Icm systems from the pathogens Legionella and Coxiella and IncI conjugation systems, which the Dot/Icm systems are believed to have evolved from.

Legionella pneumophila, a ubiquitous organism in the environment, can remarkably replicate in protozoa and within alveolar macrophages inside human lungs. In the latter case, unrestricted growth results in a form of pneumonia called Legionnaires’ Disease. The key to L. pneumophila’s survival and replication within these phagocytic cells is the Dot/Icm T4BSS, which exports a vast number of substrates into host cells. Although these factors are important, inactivation of most individual Dot/Icm substrates has no effect on the intracellular growth or virulence of L. pneumophila due to multiple levels of redundancy. Therefore, the Dot/Icm substrates do not represent viable targets for therapeutic intervention. In contrast, dot/icm mutants are fully avirulent as the Dot/Icm system is essential for allowing L. pneumophila to survive and replicate within host cells. This makes the Dot/Icm T4BSS a prime virulence target for drug development against Legionella species. However, even after twenty years of research, we have only a rudimentary understanding of how the Dot/Icm system is assembled and we know little about the secretion process.

Recently, there have been several major advances involving the Dot/Icm T4BSS. First, we demonstrated that the L. pneumophila T4BSS is located at both ends of the bacteria and it secretes substrates into host cells in a polar fashion. In addition, we showed that non-polar secretion is ineffective for proper targeting of the LCV (Legionella containing vacuole) within host cells. Second, we detected a novel interaction between DotL, which is the type IV coupling protein for the Dot/Icm system, and IcmS/IcmW (a dedicated specialized secretion chaperone pair) that is critically required for the export of a vast array of Dot/Icm substrates. This information was then used by crystallographers to obtain the structure of DotLc (the c-terminus of DotL) bound to IcmS/IcmW. Finally, we obtained the first cryoEM image of the L. pneumophila Dot/Icm apparatus.

However, typical of many advances, these discoveries have raised numerous exciting questions including: 1) how is the Dot/Icm system targeted to the poles, 2) what is the function of the DotL:chaperone complex, 3) how is the entire secretion system assembled and 4) how does it function on a molecular level? Therefore, this proposal will exploit these recent developments and is based on the hypothesis that the key to understanding L. pneumophila virulence is to determine how the Dot/Icm T4BSS assembles and functions. Our overall approach employs a multi-disciplinary strategy to examine three independent, but interconnected, aspects of the L. pneumophila Dot/Icm type IVB secretion system.

Project#1. Polar localization of the Legionella Dot/Icm T4SS.

Project#2. Role of the T4SS chaperones found in the Legionella type IV coupling protein (LT4CP) subcomplex.

Project#3. Global structural analysis of the Legionella Dot/Icm T4SS by cryo-electron microscopy (cryoEM).